It is commonly known that many small molecules will not elicit an antibody response by themselves but, when coupled to an appropriate immunogenicity conferring carrier molecule (to become an immunogen), antibodies can be prepared against the hapten. This technology is discussed in many textbooks. See Erlanger, B. F. in Methods of Enzymology, 70:85-105 (Academic Press 1980); and Hurn, B. A. L., et al., in Methods of Enzymology, 70:105- (Academic Press 1980).
Many methods of adding haptens to oligonucleotide probes are known in the literature. A review of such conjugate literature is found in Goodchild, Bioconjugate Chemistry, 1(3):165-187 (1990). Enzo Biochemical (New York) and Clontech (Palo Alto) both have described and commercialized probe labeling techniques, including techniques for labeling probes with biotin or similar haptens. In addition, co-pending applications U.S. Ser. Nos. 625,566, filed Dec. 11, 1990 and 630,908, filed Dec. 20, 1990 teach methods for labeling probes at their 5' and 3' ends respectively. The entire disclosures of the aforementioned copending applications are incorporated by reference. The hapten label or "hook" may be used either to isolate a desired target sequence (i.e.. by hybridization with a haptenated oligonucleotide and collection of the haptens with a specific binding partner); or to attach a detectable signaling moiety to a target sequence (e.g. by probing target with a haptenated oligonucleotide and using an anti-hapten conjugate with a detectable signal generating compound such as a fluorophore, chemilumiphore, colloidal particle or enzyme).
According to one known method for labeling an oligonucleotide, a label-phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis. For example, see Thuong, N. T. et al., Tet. Letters, 29(46):5905-5908 (1988); or Cohen, J. S. et al., U.S. patent application Ser. No. 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688) (1989). However, DNA synthesis reaction conditions are quite severe (e.g. iodine oxidation and ammonium hydroxide cleavage) and many haptens (e.g. biotin and fluorescein) do not readily withstand these conditions without modification. In another approach useful for labile haptens, a linker having a protected terminal amine is attached to the desired end of the oligonucleotide. The amine can be deprotected and, under milder conditions, reacted with a label.
Automated synthesis of oligonucleotides (See e.g. Beaucage and Caruthers, Tet. Letters, 22(20):1859-1862 (1981) and U.S. Pat. Nos. 4,973,679 and 4,458,066) is often the most efficient method of preparing probes. However, the hostile conditions required during automated synthesis limit the choice of labels available for labeling by this method. The present invention overcomes these drawbacks by describing novel haptens which will withstand the rather rigorous conditions of DNA synthesis. Thus, using the haptens of the invention, an oligonucleotide can be directly labeled during automated synthesis, without involving an intervening isolation or a secondary labeling reaction.
The invention has a further advantage in that successfully labeled oligonucleotides can easily be isolated from unlabeled oligonucleotides by an affinity separation method using a specific binding partner, e.g. an antibody, for the hapten.
Methodology for preparing tracer molecules also is known. For example, fluorescence polarization assays require tracers comprising an analyte-hapten coupled to a fluorescent molecule. Typically, the analyte-hapten and a known amount of tracer are allowed to compete for a limited amount of a specific binding member for the hapten, and the labeled tracer is thereby partitioned between a bound and free form. The signal from the bound form is differentiable from the signal from the free form, so that the amount of analyte-hapten can be estimated. One method for differentiating the signals is by fluorescence polarization immunoassay (FPIA), in which the "millipolarization", the "span" or the "relative intensity" can be measured as described in the literature and below. The technique of FPIA has been described, for example, in Jolley, M. E., J. Analyt. Toxicol., 5:236-240 (1981) and in Blecka, L. J. Amer. Assoc. Clin. Chem. pp. 1-6 (March 1983) ,the entire disclosures of which are incorporated herein by reference.
Some researchers have prepared antibodies to (i.e. demonstrated immunogenicity of) polyhalogenated dibenzofurans in the context of toxic byproduct detection and cleanup. For example, Vanderlaan, et al have worked with polyhalogenated p-dioxins (which differ from dibenzofuran in having a six-member, 2 oxygen spacer ring between the two benzene rings, instead of a 5 member, one oxygen furan) and polyhalogenated dibenzofurans; see e.g. U.S. Pat. No. 4,798,807, EP-A-332 819, Chemosphere, 16(8-9): 1635-39 (1987), Toxicology, 45(3):229-43 (1987). U.S. Pat. No. 4,238,472 to Albro, et al., describes antibodies to and immunogens made from similar polychlorinated compounds. Albro, et al. show that antisera to the polychlorinated dioxins and dibenzofurans had very little reactivity with the unchlorinated species.
Pandey, et al., J. Immunol. Methods, 94(1-2):237-46 (1986), describe antibodies to and immunogens made from 3-azido-N-ethylcarbazole.
However, applicants are unaware of any art demonstrating the antigenicity or immunogenicity of the claimed carbazole and dibenzofuran derivatives.